Arabidopsis CURLY LEAF functions in leaf immunity against fungal pathogens by concomitantly repressing SEPALLATA3 and activating ORA59

Arabidopsis non-host resistance against non-adapted fungal pathogens including Colletotrichum fungi consists of pre-invasive and post-invasive immune responses. Here we report that non-host resistance against non-adapted Colletotrichum spp. in Arabidopsis leaves requires CURLY LEAF (CLF), which is critical for leaf development, flowering and growth. Microscopic analysis of pathogen behavior revealed a requirement for CLF in both pre- and post-invasive non-host resistance. The loss of a functional SEPALLATA3 (SEP3) gene, ectopically expressed in clf mutant leaves, suppressed not only the defect of the clf plants in growth and leaf development but also a defect in non-host resistance against the non-adapted Colletotrichum tropicale. However, the ectopic overexpression of SEP3 in Arabidopsis wild-type leaves did not disrupt the non-host resistance. The expression of multiple plant defensin (PDF) genes that are involved in non-host resistance against C. tropicale was repressed in clf leaves. Moreover, the Octadecanoid-responsive Arabidopsis 59 (ORA59) gene, which is required for PDF expression, was also repressed in clf leaves. Notably, when SEP3 was overexpressed in the ora59 mutant background, C. tropicale produced clear lesions in the inoculated leaves, indicating an impairment in non-host resistance. Furthermore, ora59 plants overexpressing SEP3 exhibited a defect in leaf immunity to the adapted Colletotrichum higginsianum. Since the ora59 plants overexpressing SEP3 did not display obvious leaf curling or reduced growth, in contrast to the clf mutants, these results strongly suggest that concomitant SEP3 repression and ORA59 induction via CLF are required for Arabidopsis leaf immunity to Colletotrichum fungi, uncoupled from CLF’s function in growth and leaf development.     

Heteroblasty in the moss,Aphanoregma patens (Physcomitrella patens), results from progressive modulation of a single fundamental leaf developmental programme

Abstract

Leaves at the apex of a mature Aphanoregma patens (Hedw.) Lindb. (Physcomitrella patens (Hedw.) Bruch Schimp. in B.S.G.) gametophore differ markedly in size and form from those at its base. To determine how these differences are produced during development, we first examined qualitative and quantitative differences between successive leaves along the stem and among leaves at different developmental stages. Differences between successive leaves were slight and cumulative. Local changes in cell number and size combined to produce a regularly shaped and approximately bilaterally symmetrical leaf suggesting that cell division and cell expansion are regionally regulated and coordinated at the organ level. The midrib and marginal teeth are discrete characters, which were prefigured by changes in cell shape in leaves that lacked these characters. In leaf primordia, cell proliferation was responsible for most of the changes in leaf form and size early in development and may have continued as cell expansion took over as the primary contributor to leaf growth and morphogenesis. Thus, leaf heteroblasty in Physcomitrella probably results from modulation of a single developmental programme by external and/or internal forces, which alter progressively in intensity as a gametophore grows. We applied exogenous cytokinin and auxin separately to growing cultures to explore their effects on leaf growth. Cytokinin and auxin stimulated leaf cell division and leaf cell elongation, respectively. Also, young upper leaves of gametophores exposed to exogenous auxin closely resembled basal leaves of untreated plants. Therefore, endogenous cytokinins and auxins may be among the modulating internal forces involved in leaf morphogenesis and the establishment of leaf heteroblasty.     

Ultraviolet-B radiation reduces the rates of cell division and elongation in the primary leaf of wheat (Triticum aestivum L. cv Maris Huntsman)

The primary leaf of wheat (Triticum aestivum L. cv Maris Huntsman) was used as a model system to examine how elevated ultraviolet‐B (UV‐B; λ= 280–320 nm) radiation affected growth. A reduction in the rate and duration of growth of the primary leaf, in response to UV‐B, was the result of changes in both the rate and extent of cell division and elongation. UV‐B reduced the proportion of mitotically active cells (mitotic index) and increased the time taken for cell division (cell doubling time). Thus the supply of cells into the elongation zone was reduced, and this, coupled to a reduction in the rate of elongation, resulted in reduced leaf growth. This analysis of the spatial distribution of growth provided a means of calculating the age of cells within the leaves. Cells of UV‐B‐treated leaves were found to age more quickly than those of the controls. This analysis will enable future studies to take account of age‐related changes when interpreting the response of plants to any number of environmental stresses that affect leaf development.     

Early stages of leaf development in <Emphasis Type="Italic">has</Emphasis> mutant of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The elucidation of molecular mechanisms underlying the leaf development can be facilitated by the detailed anatomical study of leaf development mutants. We present an analysis of leaf anatomy and morphogenesis during early developmental stages in has mutant of Arabidopsis thaliana. The recessive has mutation affects a number of aspects in plant development, including the shape and size of both cotyledons and leaves. The earliest developmental observations suggest almost synchronous growth of the first two leaf primordia of has mutant. No significant disruption of the cell division pattern in the internal tissue is observed at the earliest stages of development, with the major anatomical difference compared to wild type primordia being the untimely maturation of mesophyll tissue cells in has mutant. At the stage of leaf blade formation, structure disruption becomes clearly evident, by irregular arrangement of the cell layers and the lack of polarity in juvenile has leaves. One distinguishing feature of the mutant leaf anatomy is the absence of mesophyll tissue differentiation. Altered has mutant leaf morphology could be at least partially accounted for by the ectopic STM activity that was found at the base of leaf primordia during early stages of leaf development in has plants.     

Abnormal cell divisions in leaf primordia caused by the expression of the rice homeobox geneOSH1 lead to altered morphology of leaves in transgenic tobacco

Transgenic tobacco plants were generated carrying a rice homeobox gene,OSH1, controlled by the promoter of a gene encoding a tobacco pathogenesis-related protein (PR1a). These lines were morphologically abnormal, with wrinkled and/or lobed leaves. Histological analysis of shoot apex primordia indicated arrest of lateral leaf blade expansion, often resulting in asymmetric and anisotropic growth of leaf blades. Other notable abnormalities included abnormal or arrested development of leaf lateral veins. Interestingly,OSH1 expression was undetectable in mature leaves with the aberrant morphological features. Thus,OSH1 expression in mature leaves is not necessary for abnormal leaf development. Northern blot and in situ hybridization analyses indicate thatPR1a-OSH1 is expressed only in the shoot apical meristem and in very young leaf primordia. Therefore, the aberrant morphological features are an indirect consequence of ectopicOSH1 gene expression. The only abnormality observed in tissues expressing the transgene was periclinal (rather than anticlinal) division in mesophyll cells during leaf blade initiation. This generates thicker leaf blades and disrupts the mesophyll cell layers, from which vascular tissues differentiate. TheOSH1 product appears to affect the mechanism controlling the orientation of the plane of cell division, resulting in abnormal periclinal division of mesophyll cell, which in turn results in the gross morphological abnormalities observed in the transgenic lines.     

Mechanisms of leaf tooth formation in Arabidopsis

Serration found along leaf margins shows species‐specific characters. Whereas compound leaf development is well studied, the process of serration formation is largely unknown. To understand mechanisms of serration development, we investigated distinctive features of cells that could give rise to tooth protrusion in the simple‐leaf plant Arabidopsis. After the emergence of a tooth, marginal cells, except for cells at the sinuses and tips, started to elongate rapidly. Localized cell division seemed to keep cells at the sinus smaller, rather than halt cell elongation. As leaves matured, the marginal cell number between teeth became similar in any given tooth. These results suggest that teeth are formed by repetition of an unknown mechanism that spatially monitors cell number and regulates cell division. We then examined the role of CUP‐SHAPED COTYLEDON 2 (CUC2) in serration development. cuc2‐3 forms fewer hydathodes and auxin maxima, visualized by DR5rev::GFP, at the leaf margin, suggesting that CUC2 patterns serration through the regulation of auxin. In contrast to a previous interpretation, comparison of leaf outlines revealed that CUC2 promotes outgrowth of teeth rather than suppression of growth at the sinuses. We found that mutants with increased CUC2 expression form ectopic tissues and mis‐express SHOOT MERISTEMLESS (STM) at the sinus between the enhanced teeth. Similar but infrequent STM expression was found in the wild type, indicating STM involvement in the serration of simple leaves. Our study provides insights into the morphological and molecular mechanisms for leaf development and tooth formation, and highlights similarities between serration and compound leaf development.     

Promotion of leaf sheath growth by gibberellic acid in a dwarf mutant of rice

The mechanism of gibberellin (GA)-induced leaf sheath growth was examined using a dwarf mutant of rice (Oryza sativa L. cv. Tan-ginbozu) treated in advance with an inhibitor of GA biosynthesis. Gibberellic acid (GA₃) enhanced the growth of the second leaf sheath, but auxins did not. Measurement of the mitotic index and cell size revealed that cell elongation rather than cell division is promoted by GA₃. Gibberellic acid increased the extensibility of cell walls in the elongation zone of the leaf sheath. It also increased the total amount of osmotic solutes including sugars in the leaf sheath, but did not increase the osmotic concentration of the cell sap, due to an accompanying increase in cell volume by water absorption. In the later stage of GA₃-induced growth, starch granules completely disappeared from leaf sheath cells, whereas dense granules remained in control plants. These findings indicate that GA enhances cell elongation by increasing wall extensibility, osmotic concentration being kept unchanged by starch degradation. Received: 28 August 1997 / Accepted: 16 October 1997     

Epidermal Cell Division and the Coordination of Leaf and Tiller Development

Initiation and development of grass leaves and tillers are oftendescribed individually with little attention to possible interrelationshipsamong organs. In order to better understand these interrelationships,this research examined epidermal cell division during developmentaltransitions at the apical meristem of tall fescue (Festuca arundinaceaSchreb.). Ten seedlings were harvested each day for a 9-d period,and lengths of main shoot leaves and primary tillers were measured.In addition, numbers and lengths of epidermal cells were determinedfor 0·5 mm segments along the basal 3 mm of each leafand tiller. Primordia development and onset of rapid leaf elongationwere characterized by an increase in the number of cells perepidermal file with mean cell length remaining near 20 µmper cell. After the leaf had lengthened to 1-1·5 mm,cells near the leaf tip ceased dividing and increased in length,at which time leaf elongation rate increased rapidly. Liguleformation, marking the boundary between blade and sheath cells,occurred prior to leaf tip emergence above the whorl of oldersheaths, while the earliest differentiation between blade andsheath cells probably began when leaves were < 1 mm long.Major transitions in leaf and tiller development appeared tobe synchronized among at least three adjacent nodes. At theoldest node, cessation of cell division in the leaf sheath wasaccompanied by initiation of cell division and elongation inthe associated tiller bud. At the next younger node the ligulewas being initiated, while at the youngest node cell divisioncommenced in the leaf primordium, as elongation of a new leafblade began. This synchronization of events suggests a key rolefor the cell division process in regulating leaf and tillerdevelopment.Copyright 1994, 1999 Academic Press Festuca arundinacea Schreb., tall fescue, cell division, leaf initiation, tillering, ligule development     

Coordination of cell proliferation and cell expansion in the control of leaf size in Arabidopsis thaliana

Size is an important parameter in the characterization of organ morphology and function. To understand the mechanisms that control leaf size, we previously isolated a number of Arabidopsis thaliana mutants with altered leaf size. Because leaf morphogenesis depends on determinate cell proliferation, the size of a mature leaf is controlled by variation in cell size and number. Therefore, leaf-size mutants should be classified according to the effects of the mutations on the cell number and/or size. A group of mutants represented by angustifolia3/grf-interacting factor1 and aintegumenta exhibits an intriguing cellular phenotype termed compensation: when the leaf cell number is decreased due to the mutation, the leaf cell size increases, leading to compensation in leaf area. Several lines of genetic evidence suggest that compensation is probably not a result of the uncoupling of cell division from cell growth. Rather, the evidence suggests an organ-wide mechanism that coordinates cell proliferation with cell expansion during leaf development. Our results provide a key, novel concept that explains how leaf size is controlled at the organ level.     

Sterols are required for cell‐fate commitment and maintenance of the stomatal lineage in Arabidopsis

Asymmetric cell division is important for regulating cell proliferation and fate determination during stomatal development in plants. Although genes that control asymmetric division and cell differentiation in stomatal development have been reported, regulators controlling the process from asymmetric division to cell differentiation remain poorly understood. Here, we report a weak allele (fk–J3158) of the Arabidopsis sterol C–14 reductase gene FACKEL (FK) that shows clusters of small cells and stomata in leaf epidermis, a common phenomenon that is often seen in mutants defective in stomatal asymmetric division. Interestingly, the physical asymmetry of these divisions appeared to be intact in fk mutants, but the cell‐fate asymmetry was greatly disturbed, suggesting that the FK pathway links these two crucial events in the process of asymmetric division. Sterol profile analysis revealed that the fk–J3158 mutation blocked downstream sterol production. Further investigation indicated that cyclopropylsterol isomerase1 (cpi1), sterol 14α–demethylase (cyp51A2) and hydra1 (hyd1) mutants, corresponding to enzymes in the same branch of the sterol biosynthetic pathway, displayed defective stomatal development phenotypes, similar to those observed for fk. Fenpropimorph, an inhibitor of the FK sterol C–14 reductase in Arabidopsis, also caused these abnormal small‐cell and stomata phenotypes in wild‐type leaves. Genetic experiments demonstrated that sterol biosynthesis is required for correct stomatal patterning, probably through an additional signaling pathway that has yet to be defined. Detailed analyses of time‐lapse cell division patterns, stomatal precursor cell division markers and DNA ploidy suggest that sterols are required to properly restrict cell proliferation, asymmetric fate specification, cell‐fate commitment and maintenance in the stomatal lineage cells. These events occur after physical asymmetric division of stomatal precursor cells.     

The mutants compacta ?hnlich, Nitida and Grandiflora define developmental compartments and a compensation mechanism in floral development in Antirrhinum majus

In order to improve our understanding of floral size control we characterised three mutants of Antirrhinum majus with different macroscopic floral phenotypes. The recessive mutant compacta ?hnlich has smaller flowers affected mainly in petal lobe expansion, the dominant mutant Grandiflora has overall larger organs, whilst the semidominant mutation Nitida exhibits smaller flowers in a dose-dependent manner. We developed a cell map in order to establish the cellular phenotypes of the mutants. Changes in organ size were both organ- and region-specific. Nitida and compacta ?hnlich affected cell expansion in proximal and distal petal regions, respectively, suggesting differential regulation between petal lobe regions. Although petal size was smaller in compacta ?hnlich than in wild type, conical cells were significantly bigger, suggesting a compensation mechanism involved in petal development. Grandiflora had larger cells in petals and increased cell division in stamens and styles, suggesting a relationship between genes controlling organ size and organ identity. The level of ploidy in petals of Grandiflora and coan was found to be equivalent to wild type petals and leaves, ruling out an excess of growth via endoreduplication. We discuss our results in terms of current models about control of lateral organ size.     

Brassinolide affects the rate of cell division in isolated leaf protoplasts of Petunia hybrida

Brassinosteroids are known to promote cell elongation in a wide range of plant species but their effect on cell division has not been as extensively studied. We examined the effect of brassinolide on the kinetics and final division frequencies of regenerating leaf mesophyll protoplasts of Petunia hybrida Vilm v. Comanche. Under optimal auxin and cytokinin conditions, 10–100 nM brassinolide accelerated the time of first cell division by 12 h but had little effect on the final division frequencies after 72–120 h of culture. One micromolar brassinolide showed the same acceleration of first cell division but inhibited the final division frequency by approximately 20%. Under sub-optimal auxin conditions, 10–100 nM brassinolide both accelerated the time of first cell division and dramatically increased the 72- to 120-h final division frequencies. Isolated protoplasts may provide a useful model system to investigate the molecular mechanisms of brassinosteroid action on cell proliferation. Received: 1 December 1997 / Revision received: 13 February 1998 / Accepted: 24 April 1998     

The maize disorganized aleurone layer 1 and 2 ( dil1, dil2) mutants lack control of the mitotic division plane in the aleurone layer of developing endosperm

The maize (Zea mays L.) endosperm consists of an epidermal like layer of isodiametric aleurone cells surrounding a central body of starchy endosperm cells. In disorgal1 (dil1) and disorgal2 (dil2) mutants the control of the mitotic division plane is relaxed or missing, resulting in mature grains with disorganized aleurone layers. In addition to orientation of the division plane, both the shape and size of the aleurone cells are affected, and often more than one layer of aleurone cells is present. Homozygous dil1 and dil2 grains are shrunken due to reduced accumulation of starchy endosperm and premature developmental arrest of the embryo, and mature mutant grains germinate at a very low rate and fail to develop into plants. However, homozygous mutant plants can be obtained through embryo rescue, revealing that both mutants have an irregular leaf epidermis as well as roots with a strongly reduced number of root hairs and aberrant root hair morphology. Our results suggest the presence of common regulatory mechanisms for the control of cell division orientation in the aleurone and plant epidermis.Abbreviations DAP days after pollination - dek defective kernel mutant - dil disorganized aleurone layer mutant - GUS -glucuronidase - LM light microscopy - PPB pre-prophase band - SEM scanning electron microscopy - TUSC Trait Utility System for Corn     

CCR1, an enzyme required for lignin biosynthesis in Arabidopsis,mediates cell proliferation exit for leaf development

After initiation, leaves first undergo rapid cell proliferation. During subsequent development, leaf cells gradually exit the proliferation phase and enter the expansion stage, following a basipetally ordered pattern starting at the leaf tip. The molecular mechanism directing this pattern of leaf development is as yet poorly understood. By genetic screening and characterization of Arabidopsis mutants defective in exit from cell proliferation, we show that the product of the CINNAMOYL CoA REDUCTASE (CCR1) gene, which is required for lignin biosynthesis, participates in the process of cell proliferation exit in leaves. CCR1 is expressed basipetally in the leaf, and ccr1 mutants exhibited multiple abnormalities, including increased cell proliferation. The ccr1 phenotypes are not due to the reduced lignin content, but instead are due to the dramatically increased level of ferulic acid (FeA), an intermediate in lignin biosynthesis. FeA is known to have antioxidant activity, and the levels of reactive oxygen species (ROS) in ccr1 were markedly reduced. We also characterized another double mutant in CAFFEIC ACID O‐METHYLTRANSFERASE (comt) and CAFFEOYL CoA 3‐O‐METHYLTRANSFERASE (ccoaomt), in which the FeA level was dramatically reduced. Cell proliferation in comt ccoaomt leaves was decreased, accompanied by elevated ROS levels, and the mutant phenotypes were partially rescued by treatment with FeA or another antioxidant (N‐acetyl‐l ‐cysteine). Taken together, our results suggest that CCR1, FeA and ROS coordinate cell proliferation exit in normal leaf development.     

The SCHIZOID gene regulates differentiation and cell division in Arabidopsis thaliana shoots

 Cell division and cell differentiation are key processes in shoot development. The Arabidopsis thaliana (L.) Heynh. SCHIZOID (SHZ) gene appears to influence cell differentiation and cell division in the shoot. The shz-2 mutant is notable in that distinct phenotypes develop, depending on the environment in which the plants are grown. When shz-2 mutants are grown in petri dishes, callus develops from the petiole and hypocotyl. In contrast, when the mutants are grown on soil, shoots appear externally stunted with malformed leaves. However, detailed examination of soil-grown mutants shows that the two phenotypes are related. Soil-grown mutants form adventitious meristems, produce a large amount of vascular tissues and have aberrant cell divisions in the meristem. Cells with abnormal cell-division patterns were found in the apical and vascular meristems, suggesting SHZ influences cell division. Development of callus in petri dishes, development of adventitious meristems and aberrations in leaves on soil suggest that SHZ influences cell differentiation. The distinct, but related phenotypes on soil and in petri dishes suggests that SHZ normally functions to regulate differentiation and/or cell division in a manner that is responsive to environmental conditions. Received: 30 July 1999 / Accepted: 22 September 1999